Elicitation of T-cell effector responses requires signal transduction through the T-cell antigen receptor (TCR), a protein complex that binds antigenic peptide presented by MHC, as well as through costimulatory receptors such as CD28. The effector responses generated from TCR signal transduction differ across individual T-cell subsets that are classified according to the expression of cell surface molecules (Smith-Garvin et al., 2009, Annu Rev Immunol 27:591-619). Expression of the surface molecule CD8, for instance, identifies a subset of T cells that respond to antigenic peptides presented in the binding groove of MHC class I. CD8+ T cells are responsible for the recognition and elimination of cells that express antigens derived from intracellular pathogens, such as viruses and intracellular bacteria, and also mutated or embryonic proteins generated by cells that have undergone malignant transformation. Although the extent to which CD8+ T cells are capable of controlling the development and progression of tumorigenesis remains uncertain, it is clear that deficiency of these cells increases the potential for the development of malignancy and that enhanced function of these CD8+ T cells can impart robust antitumor responses in both animal model systems and patients (Turcotte & Rosenberg, 2011, Adv Surg 45:341-60; June, 2007, J Clin Invest 117:1466-76). It is also clear in a number of models that although there may be an initial, potent CD8+ T-cell response, this response is often insufficient to fully protect from tumors (Schreiber et al., 2011, Science 331:1565-70). Mechanisms underlying this failure include (i) the lack of specific antigens with sufficient avidity for the TCR expressed by tumors, (ii) the absence of costimulatory ligands expressed by antigen-presenting cells (APC) within tumor-draining lymph nodes, and (iii) direct suppression of T-cell responses within the tumor microenvironment mediated by inhibitory secreted factors such as TGFβ, prostaglandin E (PGE)-2, or adenosine, as well as inhibitory cells, such as regulatory T cells (Quezada et al., 2011, Immunol Rev 241:104-18).
The potential for effective responses by CD8+ T cells in some instances of incurable malignancies, such as metastatic melanoma, has led to significant interest in defining ways to manipulate these cells to generate more potent responses as well as responses against a more diverse array of tumors. One promising approach has focused on engineering T cells to express chimeric antigen receptors (CAR). CARs are transmembrane fusion proteins that consist of an extracellular antibody domain capable of binding to a specific tumor antigen coupled to intracellular signaling domains from TCR and costimulatory components (Milone et al., 2009, Mol Ther 17:1453-64). In principle, CARs provide several advantages over the endogenous receptors of T cells. First, the engineered ligand-binding segment of CARs arises from an antibody, obviating the need for MHC presentation. Second, the antibody-binding component of the CAR can be chosen to be both specific and highly sensitive to antigens expressed selectively by tumor cells, increasing avidity of the T cell-tumor interaction and minimizing the potential for destruction of normal “bystander” host cells. Third, engagement of the CAR by ligand stimulates both TCR and costimulatory signaling modules, eliminating a requirement for expression of costimulatory ligands by tumor-draining APCs. CAR-expressing T cells that come into contact with tumor cells expressing the antigen of interest have been shown to develop functional responses that lead to tumor cell lysis and cytokine production.
There has been considerable success in the use of CARs in animal models (Milone et al., 2009, Mol Ther 17:1453-64; Dower et al., 2000, Nat Immunol 1:317-21), and recently, CAR-expressing T cells have been shown to be effective in patients to treat refractory chronic lymphocytic leukemias (CLL) (Chiang et al., 2007, J Clin Invest 117:1029-36; Loeser et al., 2007, J Exp Med 204:879-91). Although T cells engineered to express CARs are capable of overcoming some limitations of the endogenous immune system to combat tumors (e.g., CARs are not MHC restricted and hence will lyse tumor cells that have downregulated MHC expression), CAR-expressing T cells still lack intrinsic programming to overcome, perhaps, the most important component that limits CD8+ T-cell antitumor responses: the inhibitory tumor microenvironment.
However, there are a number of areas that appear to be important potential limitations to the success of CARs, especially for solid tumors. A major limitation is the loss of efficacy of the infused T cells. As reported by others (Ahmadzadeh et al., 2009, Blood 114:1537-1544; Whiteside, 2004, Cancer Immunol. Immunother. 53:865-878; Zippelius et al., 2004, Cancer Res. 64:2865-2873; Bronte et al., 2005, J. Exp. Med. 201:1257-1268; Prinz et al., 2012, J. Immunol. 188:5990-6000; Monu et al., 2007, Cancer Res. 67:11447-11454; Janicki et al., 2008, Cancer Res. 68:2993-3000), even when the T cells successfully traffic into tumors, in many instances, they appear to be inactivated within tumors rather rapidly. The mechanisms are not fully understood, but probably involve secreted immune-inhibitory factors such as TGFβ, PGE2, and adenosine within the tumor microenvironment, interactions with inhibitory leukocytes (i.e. T-regulatory cells and myeloid suppressor cells) and/or contact with inhibitory molecules on the surface of tumor cells.
The mechanisms of T cell inactivation are not well understood, especially in CARs, where, unlike native T cells, the signal transduction mechanisms have not been well studied. Thus, there is a need in the art to develop compositions and methods for enhancing T cell activation and killing ability during adoptive T cell transfer. The present invention satisfies this unmet need.